Hundreds of nuclear-encoded proteins are required for mitochondrial metabolism, growth, division, and partitioning to daughter cells, and virtually all of these proteins must be imported into the organelle. Many of these imported proteins must cross two membranes to reach their destination inside mitochondria. Complicating matters, the inner membrane must maintain an electrochemical potential to drive the synthesis of ATP. To determine how proteins are translocated across the mitochondrial inner membrane, we have isolated and analyzed mutants in the yeast, Saccharomyces cerevisiae, defective in import. Our studies have identified two proteins of the inner membrane, Mas6p and Sms1p, that are required for the translocation of proteins into the matrix. Our recent studies suggest that proteins are translocated across the inner membrane through an aqueous channel, and that Mas6p (and possibly Sms1p) form part of this channel. Two goals of this proposal are to pinpoint the roles of Mas6p and Sms1p in import. We have found that Mas6p and Sms1p interact with each other and with additional proteins to mediate import, raising the possibility that Mas6p and Sms1p are part of a translocation complex in the inner membrane. A third goal of this proposal is to identify new members of this complex. Project 1: Determine the role of Mas6p in import. Mas6p is an essential import component that directly interact with precursors as they cross the inner membrane. Mas6p appears to have two functional domains: a hydrophilic amino-terminal region that faces the intermembrane space and a hydrophobic carboxyl-terminal domain inserted in the inner membrane. To determine the function of each part of Mas6p, we are making specific alterations in Mas6p and testing their effect on import. For example, are asking if mutations in the amino-terminal domain of Mas6p prevent the binding of precursors to the inner membrane import machinery. In addition, we are asking if the carboxyl-terminal domain forms part of a protein-translocating channel in the inner membrane. Project 2: Determine the function of Sms1p in import. Sms1p, which is also essential for import, has been shown to interact with Mas6p. To understand the role of Sms1p in import, we will generate new alleles of sms1 and examine their effects on import. For example, we will ask if sms1 mutants, like mas6 mutants, are defective in an inner membrane channel. Project 3: Identify new membrane import components and analyze their function. Using both genetic and biochemical approaches, we will identify new inner membrane import components and analyze their role in the translocation of precursors into the matrix.